The function of the nervous system is dependent on complex interactions between networks of neurons composed of multiple neuron types. Understanding how these networks function both in health and disease is dependent on understanding the precise connectivity between specific neurons types. It is therefore apparent that, in order to have an adequate understanding of the nervous system, it is necessary to have detailed descriptions of neuronal connectivity with the same level of precision at which these systems operate. The research proposed here is aimed at the development, refinement, and validation of a novel set of tools that will allow researchers to readily and systematically uncover neural circuits with cell type-specific resolution. These tools build on previous work in one of the PIs lab, developing and validating the potential for use of genetically modified rabies viruses, in combination with other genetic and viral technologies, to probe neural circuits. The new tools to be developed and tested include mouse lines and helper viruses which can be used to achieve cell type specific expression of genes that interface with the rabies tracing system. This will allow the modified rabies viruses to selectively infect specific cell types and to label the direct inputs to those cells. These new tools will be tested and protocols developed for their use in a broad range of nervous system structures, whose function is relevant to understanding disease states. New variants of rabies virus will also be generated in order to interface with the newly developed mouse lines. These variants will express genes to drive conditional expression of genes encoded in the genomes of the transgenic mice, such that inputs to specific cell types targeted for infection by the rabies virus can be identified. These new rabies viruses will also be tested and protocols developed for assaying neural circuits in a broad range of relevant structures. Overall, this project will result in the generation and validation of very valuable new tools which will then be available to the neuroscience research community. PUBLIC HEALTH RELEVANCE: Understanding neural circuits with increasingly sophisticated and higher resolution tools is crucial to understanding diseases that are caused by neural circuit disorders, including Parkinson's, neuromuscular disorders, paralysis, schizophrenia, depression, autism and attention disorders, among many others. The development of new tools for revealing circuits will therefore have a large impact on these diseases.